Compensational regulation of bHLH transcription factors in the postnatal development of BETA2/NeuroD1-null retina

Abstract

The bHLH transcriptional factor BETA2/NeuroD1 is essential for the survival of photoreceptor cells in the retina. Although this gene is expressed throughout the retina, BETA2/NeuroD1 knockout mice show photoreceptor cell degeneration only in the outer nuclear layer of the retina; other retinal neurons are not affected. Previous studies on retina explants lacking three bHLH genes revealed that retinal neurons in the inner nuclear layer require multiple bHLH genes for their differentiation and survival. However, single- or double-gene mutations show no or a lesser degree of abnormalities during eye development, likely because of compensation or cooperative regulation among those genes. Because not all null mice survive until the retina is fully organized, no direct evidence of this concept has been reported. To understand the regulatory mechanisms between bHLH factors in retinal development, we performed a detailed analysis of BETA2/NeuroD1 knockout mice. BETA2/NeuroD1 was expressed in all 3 layers of the mouse retina, including all major types of neurons. In addition, a null mutation of BETA2/NeuroD1 resulted in up-regulation of other bHLH genes, Mash1, Neurogenin2, and Math3, in the inner nuclear layer. Our data suggest that compensatory and cross regulatory mechanisms exist among the bHLH factors during retinal development.

Fig. 1

Spatiotemporal expression of BETA2/NeuroD1 in the retina. Immunoreactivity of anti-NeuroD1 antibodies at P0 (A), P3 (B), P5 (C), P10 (D), P15 (E), and P30 (F). NL, neuroblastic layer; GCL, ganglionic cell layer; ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; arrowheads, BETA2/NeuroD1-expressing neurons in the NL; arrows, BETA2/NeuroD1-expressing neurons in the INL and INL. Scale bar = 100 µm.

Fig. 2

Characterization of BETA2/NeuroD1-expressing cells in the retina. Anti-NeuroD1 double immunostaining with Calbindin (top row), PKCα (middle row), and Pax6 (bottom row) on wild-type (BETA2^+/+) retina at P15. ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglionic cell layer; arrows, double-positive cells; arrowheads, single-positive cells;. Scale bar = 50 µm.

Fig. 3

Population of major retinal neurons in the inner nuclear layer (INL) and ganglionic cell layer (GCL) of the BETA2/NeuroD1-null retina. (A) Immunohistochemical analysis with Calbindin (top row), PKCα (middle row), and Pax6 (bottom row). Scale bar = 100 µm. (B) Quantification of antibody-positive cells in the INL and GCL. (C) Total number of neurons in the INL and GCL. For B and C, data represent the means ± S.E.M. Statistical significance (P value) was determined using Student’s t-test.

Fig. 4

Up-regulation of Mash1 and Neurogenin2 expression levels in the BETA2/NeuroD1-null retina. Immunohistochemical analysis was performed with anti-Mash1, anti-Neurogenin2, and anti-Mash1 antibodies on wild-type (BETA2^+/+) and knockout (BETA2^−/−) mice at P5, P10, and P15. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglionic cell layer. Scale bar = 50 µm.

Fig. 5

Quantitative RT-PCR (qRT-PCR) analysis of Mash1, Neurogenin2, and Math3 genes in the BETA2/NeuroD1-null retina. qRT-PCR analyses were performed with retinas from four independent different groups of wild-type (BETA2^+/+) and BETA2/NeuroD1-null (BETA2^−/−). Data represent the mean ± S.D. from four independent experiments (*P < 0.05, Student's t test).