Two transcription factors, Pou4f2 and Isl1, are sufficient to specify the retinal ganglion cell fate

Abstract

As with other retinal cell types, retinal ganglion cells (RGCs) arise from multipotent retinal progenitor cells (RPCs), and their formation is regulated by a hierarchical gene-regulatory network (GRN). Within this GRN, three transcription factors--atonal homolog 7 (Atoh7), POU domain, class 4, transcription factor 2 (Pou4f2), and insulin gene enhancer protein 1 (Isl1)--occupy key node positions at two different stages of RGC development. Atoh7 is upstream and is required for RPCs to gain competence for an RGC fate, whereas Pou4f2 and Isl1 are downstream and regulate RGC differentiation. However, the genetic and molecular basis for the specification of the RGC fate, a key step in RGC development, remains unclear. Here we report that ectopic expression of Pou4f2 and Isl1 in the Atoh7-null retina using a binary knockin-transgenic system is sufficient for the specification of the RGC fate. The RGCs thus formed are largely normal in gene expression, survive to postnatal stages, and are physiologically functional. Our results indicate that Pou4f2 and Isl1 compose a minimally sufficient regulatory core for the RGC fate. We further conclude that during development a core group of limited transcription factors, including Pou4f2 and Isl1, function downstream of Atoh7 to determine the RGC fate and initiate RGC differentiation.

Keywords: cell fate specification; gene regulation; neural development; retinal development; transcription factors.

Fig. 1.

Isl1 and Pou4f2 specify RGC fate. (A–D) Immunostaining for Pou4f2 (red) on E14.5 Atoh7^tTA/+ (A), Atoh7^tTA/tTA (B), P&I^EE;Cre (C), and P&I^EE (D) retinal sections. (Scale bar, 37.5 µm.) (A′–D′) Immunostaining for Isl1 (green) on E14.5 retinal sections of the different genotypes. (A′′–D′′) Merged images of Pou4f2 and Isl1 staining of retinal sections with the different genotypes. Note there are more Pou4f2- and Isl1-expressing cells in the NBL of the P&I^EE retina (D′′) than in the NBL of the Atoh7^tTA/+ retina (A′′). (E) Counting of Pou4f2⁺ and Isl1⁺ cells in the NBL and GCL. *P < 0.01 (compared with Atoh7^tTA/+) as determined by Student’s t test. Error bars indicate ± SD.

Fig. 2.

Ectopic Pou4f2 and Isl1 activate the endogenous Pou4f2 and Isl1 genes. (A–D) Immunostaining for Pou4f2 (red) in Atoh7^tTA/+ (A), Atoh7^tTA/tTA (B), P&I^EE;Cre (C), and P&I^EE (D) retinal sections at E17.5. (Scale bar, 75 µm.) (A′–D′) Immunostaining for Isl1 (green) of retinal sections with the different genotypes. (A′′–D′′) Merged images of Pou4f2 and Isl1 staining of retinal sections from the different genotypes. (E) Detection of different mRNA transcripts by RT-PCR from E17.5 retinal tissues of the different genotypes as indicated.

Fig. 3.

Isl1 and Pou4f2 promotes cell-cycle exit. (A) Immunostaining of BrdU (green) and Pou4f2 (red) on Atoh7^tTA/+ retinal sections at E14.5. (Scale bar, 150 µm.) (A′ –A′′′) High-magnification images of A in separate (red for Pou4f2 and green for BrdU) and merged channels. (Scale bar, 25 µm.) (B) Costaining of BrdU and Pou4f2 on E14.5 Atoh7^tTA/tTA retinal sections. (B′–B′′′) High-magnification images of B in separate and merged channels. (C) Costaining of BrdU and Pou4f2 on E14.5 P&I^EE retinal sections. (C′–C′′′) High-magnification images of C in separate and merged channels.

Fig. 4.

The gene-expression program in the RGCs of the P&I^EE retina is almost fully activated. Immunostaining for Nflm (green), PGP9.5 (green), Onecut1 (green), and Pou4f1 (red) and in situ hybridization for Sncg, Nflm, Rbpms, Stmn2, Sox4, Sox11, Ina, Eya2, Cyclin D1 (Ccnd1), and Gli1 in Atoh7^tTA/+, Atoh7^tTA/tTA, and P&I^EE retinal sections at E14.5. With the exception of Eya2, these genes are fully activated in the P&I^EE retina. (Scale bar, 150 µm.)

Fig. 5.

RGCs in the P&I^EE retina survive to postnatal stages. (A) The optic nerves from P18 Atoh7^tTA/+, Atoh7^tTA/tTA, P&I^EE;Cre, and P&I^EE retinas. (B–E) Whole-mount immunostaining of RGC axons with SMI32 in Atoh7^tTA/+, Atoh7^tTA/tTA, P&I^EE;Cre, and P&I^EE retinas at P18. (Scale bar, 150 µm.) (F–I) Immunolabeling of RGCs with anti-Pou4f1 in whole-mount retinas of the various genotypes at P18. (Scale bar, 75 µm.)

Fig. 6.

RGC subtypes form in the P&I^EE retina. (A–C) Immunostaining for Tbr2 (green) and Pou4f2 (red) in E14.5 Atoh7^tTA/+ (A), Atoh7^tTA/tTA (B), and P&I^EE (C) retinal sections. Arrows indicate the Tbr2⁺ cells. (Scale bar, 25 µm.) (D–F) Flat-mount immunostaining for melanopsin to visualize ipRGCs (indicated by arrows) in retinas of different genotypes at P30. (Scale bar, 75 µm.) (G–I) Staining for CART to visualize dsRGCs in retinas of different genotypes at P30. (J–L) Costaining of Calbindin (Calb, green) and Pou4f1 (red) in flat-mount retinas of the different genotypes at P30. (M) Cell counting of the different RGC subtypes in retinas with the different genotypes. Melan, melanopsin. *P < 0.01 (compared with Atoh7^tTA/+) as determined by Student’s t test. Error bars indicate ± SD.

Fig. 7.

RGCs in the P&I^EE retina are physiologically functional and show robust light-evoked spike activity. (A–E) Spike raster plots for cells recorded using both MEA and single-cell patch clamping. (A) ON-sustained cells showed sustained spike activity for the onset of light but stopped spiking at light offset. Each row shows the response of a single cell (n = 13). The light stimulus (1 s in duration) is represented by the black bar on top. The black traces show cells recorded in an MEA; the gray traces show cells recorded by single-cell patch-clamp recording. (B) ON-transient cells showed brief spike activity at light onset (n = 12). (C) OFF-sustained cells exhibited sustained activity at light offset (n = 11). (D) OFF-transient cells had phasic spike activity at light offset (n = 11). (E) ON-OFF cells showed spike activity at both light onset and offset (n = 8). (F) A window created in the GCL to record light-evoked activity in single cells. Even in this small window, a majority of cells (white marks) responded with spike activity to light. (Scale bar, 25 µm.) (G) Light-evoked spike activity of an ON transient cell in seven trials. The light response was robust and precise across trials. (H) Light-evoked EPSCs of the cell shown in G for five trials. Cells were held at Vh = −60 mV to isolate EPSCs. The responses of the trials are shown superimposed, illustrating the precision of the responses. (Inset) Sample spontaneous EPSCs evoked in the absence of light. (I) Light-evoked IPSCs of a cell shown in G for five trials (Vh = 0 mV). (Inset) Spontaneous IPSCs observed in the absence of light. (J) Comparison of peak light-evoked EPSCs in five cells and light-evoked IPSCs in three cells. Each line indicates the EPSC and IPSC recorded from the same cell.

Fig. 8.

A model for RGC fate determination in development. In this model, Atoh7 activates a small set of transcription factor genes, including Pou4f2 and Isl1, in a subset of Atoh7-expressing cells. Expression of this group of transcription factors determines the RGC fate. The question mark indicates the unidentified member(s) of this group. Once expressed, these factors can sustain their own expression, and Atoh7 becomes disposable. Arrows designating regulations between factors within the core group (including Pou4f2 and Isl1) are conceptual and do not necessarily implicate direct regulation (see text). This core group of transcription factors in turn activates the whole gene-expression program for RGC differentiation.