Figure 1.
Delayed differentiation of amacrine precursors in the Pax6loxP/loxP ;α-Cre retina.
(A) A scheme of the stages of amacrine interneuron differentiation. Amacrine cells evolve from FoxN4-expressing RPCs. In the postmitotic amacrine precursors, FoxN4 is reduced and Ptf1a expression is initiated. The Ptf1a-positive precursors migrate to the prospective INL, lose Ptf1a expression and initiate expression of TFs involved in the differentiation of amacrine subtypes (e.g. Ap2α and bHLHb5). The final differentiation of amacrine cells occurs a few days after birth with accumulation of neurotransmitters and transporters (e.g. GABA, glycine transporter GlyT1). Expression of amacrine specification and differentiation markers in control (B,C,F,G,J,K,N,O) and Pax6loxP/loxP;α-Cre OC (D,E,H,I,L,M,P,Q). Expression of FoxN4 (B–E, the inset in B and D is Crx on adjacent section) as detected using ISH. Indirect immunofluorescence (IIF) was employed to detect the expression of Ptf1a and Pax6 (green and red, respectively, F–I; adjacent sections to B-E respectively), bHLHb5 and Ap2α (green and red, respectively, J–M) and GABA (green, N–Q) during various stages of development as indicated. Pax6 (red in F–I and not shown) and Crx (inset in B,D and not shown) expression determined by IIF or ISH was used to identify the Pax6-deficient area (yellow line in D,E,H,I,L,M) and to delineate the neurogenic and nonneurogenic RPC populations in the Pax6loxP/loxP;α-Cre retina (numbered 1 and 2 and separated by a dotted white line in D and H). Abbreviations: GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; NBL, neuroblastic layer. Scale bar in C is 100 µm.
Figure 2.
Neurogenic Pax6- RPCs in the Pax6loxP/loxP ;α-Cre retina display increased cell-cycle exit and sustained expression of cell-cycle factors.
A single pulse of BrdU was administered at E14.5, E15.5, E16.5 and E18.5, 24 h prior to sacrifice as indicated (A, B). Pax6 expression was detected on adjacent sections to identify the recombination area in the Pax6loxP/loxP;α-Cre OC. (A) The percentage of BrdU + PCNA-/BrdU+Total was determined for the Pax6loxP/loxP control (green bars) and Pax6loxP/loxP ;α-Cre (blue bars) distal retina. Pax6- RPCs show increased cell-cycle exit at all stages tested (19% (SD=0.6%), 21% (SD=2.7%) and 24.1% (SD=2.6%) compared to 16.6% (SD=0.93%), 16.7% (SD=2%) and 15.9% (SD=2%) in control at E14.5, E16.5 and E18.5, respectively; p≤0.05, n≥3). (B) The number of BrdU+ and BrdU + Ki67- cells was quantified and the ratio BrdU + Ki67-/BrdU+Total was used to compare cell-cycle exit rate in control (green bar) and Pax6loxP/loxP ;α-Cre (blue bar) distal retina at E15.5. Pax6- RPCs show increased cell-cycle exit (37.3% (SD=3.6%) in Pax6- compared to 14% (SD=1.5%) in control; p<0.01, n=6 for both genotypes). Triple immunofluorescence for PCNA, Ki67 and BrdU (red, green and blue, respectively, in C–J) in control (C–F) and Pax6loxP/loxP ;α-Cre (G–J) distal retina showing mitotic PCNA + Ki67 + BrdU+ in both control and mutant (white circles in D–F,H–J) and abnormal PCNA + Ki67-BrdU- cells detected only in Pax6loxP/loxP ;α-Cre (yellow circles in H–J) OC. Scale bars in C,D are 100 and 25 µm, respectively.
Figure 3.
High-throughput analysis of transcriptional alterations in Pax6- RPCs.
(A) Scatterplot representing the fold change in gene expression in Pax6- versus control retinas (y axis) plotted against the intensity value in the control (x axis). Each spot corresponds to one gene; only genes whose expression changed at least 1.5-fold are shown. (B) Gene ontology (GO) analysis conducted using DAVID bioinformatics resource [32] for genes whose expression was altered in Pax6- versus control retinas, showing enrichment in various proliferation- and cell-cycle-related GO terms.
Figure 4.
Increased expression of cell-cycle factors in the Pax6loxP/loxP ;α-Cre retina.
Expression of Pax6 (A,E,I,M) and of cell-cycle progression and withdrawal factors was monitored using immunofluorescence in control (A–D,I–L) and Pax6loxP/loxP ;α-Cre (E–H,M–P) distal retina at E16 and E17, as indicated. Pax6- area was delineated by staining for Pax6 protein (E,M) on adjacent sections (dotted line in E–H,M,N). Expression of Ccnd2 (B,F) Ccnd3 (C,G), P57Kip2 (D,H) and P27Kip1 (green in J–L,N–P) is upregulated in most of the Pax6- RPCs of Pax6loxP/loxP ;α-Cre retina. Ccnd1 and P27Kip1, which are expressed in different cells in the control (J) are coexpressed in Pax6- RPCs (N). For the control and Pax6-deficient cells, P27Kip1-expressing cells are negative for Ki67 (red in K,O) and BrdU (red in L, P). Abbreviations: GCL, ganglion cell layer; NBL, neuroblastic layer. Scale bar in A is 100 µm.
Figure 5.
Abrogated expression of factors implicated in regulating RPC proliferation in Pax6loxP/loxP ;α-Cre retina.
The expression pattern of different factors was monitored on sections from eyes of control (A-D,I,J) and Pax6loxP/loxP ;α-Cre (E-H,K,L) mice. Nr2e1 (E12.5 A,E; E14.5 B,F) and Plagl1 (E12.5 I,K; E14.5 J,L) were detected using ISH while Vsx2 (green; E12.5 C,G; E15.5 D,H) Pax6 (red; E12.5 C,G; E15.5 D,H) were detected by IIF analysis. Pax6 (red in C,D,G,H and not shown) and Crx (inset in E and not shown) expression were used to delineate the different RPC populations in the Pax6loxP/loxP;α-Cre retina (marked with dotted line in E-H,K,L). Scale bar in F is 100 µm.
Figure 6.
Hedgehog signaling is disrupted in Pax6loxP/loxP ;α-Cre retina.
Expression of Gli1 was detected using ISH (B,D,F,H) and Pax6 protein was detected by IIF on adjacent sections (A,C,E,G) for control (A-D) and Pax6loxP/loxP ;α-Cre (E-H) OC. Gli1 expression was abrogated in all Pax6- RPCs (F,H) compared to controls (B,D) at both E14.5 (B,F) and E18.5 (D,H). Pax6- area was determined by antibody labeling on adjacent sections and marked by dotted line (E-H). Scale bar in B is 100 µm.
Figure 7.
Scheme depicting Pax6 roles during the transition from proliferating retinal progenitor to differentiating retinal neuron.
(A) In normal cycling retinal progenitors, Pax6 regulates the balance between proliferation promoting (i.e. Nr2e1, Vsx2, Hedgehog (HH) signaling) and inhibiting factors (i.e. Plagl1). These in turn regulate the expression of genes which induce either progression of (i.e. Ccnd1–3) or withdrawal (P27Kip1, P57Kip2) from the cell cycle. It is also required for the expression of bHLH proneural factors (Neurog2, Atoh7, and Ascl1) presumed to inhibit cell-cycle factors as well as promote specific retinal lineages. (B) Pax6 loss from RPCs results in aberrant cell-cycle exit as Ccnd1–3, as well as P27Kip1 and P57Kip2, are elevated and several cell-fate determination factors show reduced (bHLH proneural factors) or increased (Six3, Sox2) expression. The combined outcome of these alterations is delayed differentiation of the Pax6-deficient cells to only one subclass of retinal interneurons.