The Ath5 proneural genes function upstream of Brn3 POU domain transcription factor genes to promote retinal ganglion cell development

Abstract

During retinogenesis, the Xenopus basic helix-loop-helix transcription factor Xath5 has been shown to promote a ganglion cell fate. In the developing mouse and chicken retinas, gene targeting and overexpression studies have demonstrated critical roles for the Brn3 POU domain transcription factor genes in the promotion of ganglion cell differentiation. However, the genetic relationship between Ath5 and Brn3 genes is unknown. To understand the genetic regulatory network(s) that controls retinal ganglion cell development, we analyzed the relationship between Ath5 and Brn3 genes by using a gain-of-function approach in the chicken embryo. We found that during retinogenesis, the chicken Ath5 gene (Cath5) is expressed in retinal progenitors and in differentiating ganglion cells but is absent in terminally differentiated ganglion cells. Forced expression of both Cath5 and the mouse Ath5 gene (Math5) in retinal progenitors activates the expression of cBrn3c following central-to-peripheral and temporal-to-nasal gradients. As a result, similar to the Xath5 protein, both Cath5 and Math5 proteins have the ability to promote the development of ganglion cells. Moreover, we found that forced expression of all three Brn3 genes also can stimulate the expression of cBrn3c. We further found that Ath5 and Brn3 proteins are capable of transactivating a Brn3b promoter. Thus, these data suggest that the expression of cBrn3c in the chicken and Brn3b in the mouse is initially activated by Ath5 factors in newly generated ganglion cells and later maintained by a feedback loop of Brn3 factors in the differentiated ganglion cells.

Figure 1

Expression of Cath5 during retinogenesis as detected by in situ hybridization. (A–C) Cath5 expression is initiated at E2.5 and confined to the central retina at E3.5 in scattered cells of the ventricular zone and ganglion cell layer, and its expression continues to be strong in the central retina at E7.5. (D–F) Cath5 expression is gradually lost in the central retina from E9.5 to E14.5. (G–J) In the peripheral region, Cath5 expression is down-regulated earlier in the temporal retina than in the nasal retina at E9.5 and E13.5. GCL, ganglion cell layer; L, lens; R, retina; VZ, ventricular zone. [Bars = 56 μm (B), 50 μm (D, E, and G–J), and 25 μm (A, C, and F).]

Figure 2

Overexpression of Cath5 in retinal progenitors stimulates cBrn3c expression. (A and B) Cath5 expression was examined by in situ hybridization in sections from E11.5 retinas infected with RCAS-AP and RCAS-Cath5 viruses. (C–F) At E11.5 and E14.5, many more cells immunoreactive for cBrn3c were observed in the ventricular zone of intermediate region from retinas infected with RCAS-Cath5 viruses, compared with those infected with RCAS-AP viruses. (G–L) At E11.5, flat-mount nasal retinas infected with RCAS-AP (G, I, and K) and RCAS-Cath5 (H, J, and L) viruses from the central (G and H), intermediate (I and J), and peripheral (K and L) regions were immunostained with anti-cBrn3c antibody and photographed at the level of ventricular zone. The increase in the number of cBrn3c-immunoreactive cells displays a central-to-peripheral gradient in retinas infected with RCAS-Cath5 viruses. Arrows point to clusters of cells immunoreactive for cBrn3c. [Bar = 50 μm (E and F) and 25 μm (A–D and G–L).]

Figure 3

Effect of misexpression of Cath5 and Math5 on the expression of cBrn3c and ganglion cell production. (A) Quantitation of cBrn3c⁺ cells per section in the ventricular zone of temporal (T) and nasal (N) regions from retinas infected with RCAS-AP and RCAS-Cath5 viruses at indicated stages. Each histogram represents the mean ± SD for at least three retinas. (B and C) Quantitation of cBrn3c⁺, Islet1⁺, and NF200⁺ cells in E5.5 retinas infected with RCAS-Cath5 (B) or RCAS-Math5 (C) viruses. Misexpression of Cath5 and Math5 resulted in a significant increase in the number of cBrn3c⁺, Islet1⁺, and NF200⁺ cells in the retina. Each histogram represents the mean ± SD for at least three retinas. *, P < 0.01 by Student's t test.

Figure 4

Misexpression of Math5 and Brn3 factors stimulates cBrn3c expression. Sections from intermediate regions of E11.5 control retinas (A) and retinas infected with RCAS-Math5 (B), RCAS-Brn3a (C), RCAS-Brn3b (D), RCAS-Brn3c (E), or RCAS-Brn3cΔ8 (F) viruses were immunostained with anti-cBrn3c antibody. Misexpressed Math5, Brn3a, Brn3b, and Brn3c caused a significant increase in the number of cBrn3c immunoreactive cells in the ventricular zone, whereas misexpressed Brn3cΔ8 had no effect. (Bar = 25 μm.)

Figure 5

Transactivation of a Brn3b promoter by Ath5 and Brn3 proteins. (A) Schematic of the reporter construct in which the luciferase reporter gene (Luc) is placed under the control of a 4.6-kb Brn3b promoter sequence. (B) Relative luciferase activities after cotransfection of indicated expression plasmids with the reporter plasmid in 293 cells. Results represent the mean ± SD of triplicate assays in a single experiment.

Figure 6

Schematic illustrating regulatory relationships between Ath5 and Brn3 factors during retinal ganglion cell development. In the chicken, Cath5 may directly activate cBrn3c expression, which in turn may activate the expression of cBrn3a and cBrn3b. The maintenance of cBrn3c expression may be achieved via positive autoregulation and feedback activation by cBrn3a and cBrn3b. An analogous transcriptional cascade may control retinal ganglion cell development in the mouse except that Math5 is responsible for the initial expression of Brn3b instead of Brn3c.