Requirement of Multiple Basic Helix-Loop-Helix Genes for Retinal Neuronal Subtype Specification

Tadamichi Akagi,Tomoyuki Inoue,Goichi Miyoshi,Yasumasa Bessho,Masayo Takahashi,Jacqueline E Lee,François Guillemot,Ryoichiro Kageyama

doi:10.1074/jbc.m400871200

Tadamichi Akagi, Tomoyuki Inoue + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m400871200

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Abstract

Retinal precursor cells give rise to six types of neurons and one type of glial cell during development, and this process is controlled by multiple basic helix-loop-helix (bHLH) genes. However, the precise mechanism for specification of retinal neuronal subtypes, particularly horizontal neurons and photoreceptors, remains to be determined. Here, we examined retinas with three different combinations of triple bHLH gene mutations. In retinas lacking the bHLH genes Ngn2, Math3, and NeuroD, horizontal neurons as well as other neurons such as bipolar cells were severely decreased in number. In the retina lacking the bHLH genes Mash1, Ngn2, and Math3, horizontal and other neurons were severely decreased, whereas ganglion cells were increased. In the retina lacking the bHLH genes Mash1, Math3, and NeuroD, photoreceptors were severely decreased, whereas ganglion cells were increased. In all cases, glial cells were increased. The increase and decrease of these cells were the result of cell fate changes and cell death and seem to be partly attributable to the remaining bHLH gene expression, which also changes because of triple bHLH gene mutations. These results indicate that multiple bHLH genes cross-regulate each other, cooperatively specify neuronal subtypes, and regulate neuronal survival in the developing retina.

Highlights

Retinal precursor cells give rise to six types of neurons and one type of glial cell during development, and this process is controlled by multiple basic helix-loophelix genes
There are six types of neurons and one type of glial cell forming three cellular layers as follows: (i) rod and cone photoreceptors in the outer nuclear layer (ONL)1; (ii) horizontal, bipolar, and amacrine interneurons and Muller glia in the inner nuclear layer (INL); and (iii) ganglion and displaced amacrine cells in the ganglion cell layer (GCL)
Retinal basic helix-loophelix (bHLH) Gene Expression Is Affected by the Lack of Other bHLH Genes—It has been shown that retinal neuronal subtype specification is controlled by multiple bHLH genes such as Mash1, Ngn2, Math3, NeuroD, and Math5 (7–9, 16 –22, 35)

Summary

EXPERIMENTAL PROCEDURES

Generation of Double and Triple Mutant Mice—Math3 [21], Mash1 [28], NeuroD [29], and Ngn2 [30] mutant mice were generated previously. Mash1;NeuroD, Ngn2;NeuroD and Ngn2;Math double-mutant mice were obtained by crossing each double heterozygous mouse. Mash1;Math3;NeuroD, Mash1;Ngn2;Math, and Ngn2;Math3;NeuroD triple mutant mice were obtained by crossing each triple heterozygous mouse or triple-heterozygous female and (Mash1ϩ/Ϫ;NeuroDϩ/Ϫ, Mash1ϩ/Ϫ;Ngn2ϩ/Ϫ, or Ngn2ϩ/Ϫ;NeuroDϩ/Ϫ);Math3Ϫ/Ϫ male mouse. Retinal explants were prepared from E17.5 embryos and cultured for 4, 8, or 14 days to examine the postnatal development of the mutant retina. The digoxigenin-labeled probes used in this study were Mash1 [10], Ngn2 [12], Math3 [15], NeuroD [34], and Math5 [11]

RESULTS

Roles of Neurogenic bHLH Genes in Retinal Development

DISCUSSION