A Comprehensive Negative Regulatory Program Controlled by Brn3b to Ensure Ganglion Cell Specification from Multipotential Retinal Precursors

Abstract

The retinal ganglion cells (RGCs) are the sole output neurons in the retina that form the optic nerve and convey light signals detected by photoreceptors to the higher visual system. Their degeneration and damage caused by glaucoma and injury can lead to blindness. During retinogenesis, RGCs are specified from a population of multipotential precursors capable of generating RGC, amacrine, horizontal, and cone cells. How the RGC fate is selected from these multiple neuron fates is unknown at present. Here we show that the previously unsuspected POU domain transcription factor Brn3b (brain-specific homeobox/POU domain protein 3b) plays such a critical role. Loss of Brn3b function in mice leads to misspecification of early RGC precursors as late-born RGC, amacrine, and horizontal cells, whereas misexpressed Brn3b suppresses non-RGC cell fates but promotes the RGC fate. Microarray profiling and other molecular analyses reveal that, in RGC precursors, Brn3b normally represses the expression of a network of retinogenic factor genes involved in fate commitment and differentiation of late-born RGC, amacrine, horizontal, and cone cells. Our data suggest that Brn3b specifies the RGC fate from multipotential precursors not only by promoting RGC differentiation but also by suppressing non-RGC differentiation programs as a safeguard mechanism.

Molecular Control of Retinal Ganglion Cell Specification and Differentiation

“…Pou4f2 regulates a large set of downstream genes to fulfill its crucial function in RGC specification and differentiation. Microarray gene expression profiling of Pou4f2 wild-type and mutant retinas has revealed hundreds of genes whose expression is altered in the mutant (Mu et al, 2004;Qiu et al, 2008). Among the downregulated genes are those encoding transcription factors and proteins involved in morphogenesis, nervous system development, neuronal cell projection, synaptic vesicle, and neurofilament, consistent with the role of Pou4f2 in RGC development.…”

“…Interestingly, there seems to be always one Pou4f factor whose spatiotemporal expression pattern follows closely with the timing of RGC generation in other vertebrates, such as cPou4f3/cBrn3c and XBrn3d, in the case of chicken and Xenopus, respectively (Hutcheson and Vetter, 2001;Liu et al, 2000a). This correlative early Pou4f expression suggested to us that Pou4f function might not only be required for RGC differentiation but also for an earlier role (Xiang, 1998), which we later showed to be in RGC fate specification (Qiu et al, 2008). All three Pou4f genes were deleted in mice by gene targeting to study their developmental function Gan et al, 1999;Gan et al, 1996;McEvilly et al, 1996;Xiang et al, 1997;Xiang et al, 1996).…”

“…Pou4f2 is not only necessary but also sufficient to promote RGC differentiation from retinal progenitors. In mouse embryonic retinas, misexpressed Pou4f2 dramatically increased Pou4f1-positive RGCs (Qiu et al, 2008). When misexpressed in chick retinal progenitors by the replication-competent RCAS retroviral vector, mouse Pou4f2 increased cells immunoreactive for Isl1 and NF200, two RGC markers, by 20-50% (Fig.…”

“…RGC axon guidance errors are also present at multiple intraocular and extraocular points along their projection pathways in developing and adult Pou4f2 null mutant mice (Erkman et al, 2000). Meanwhile, inactivating Pou4f2 appears to cause many presumptive RGC precursors to switch to amacrine or horizontal cell fates (Qiu et al, 2008). These improperly differentiated cells are likely to degenerate by apoptosis as cell death significantly increases in the mutant retina (Gan et al, 1999;Xiang, 1998).…”

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

“…However, initial analysis of knockout mice (10) seem to argue against roles for Pou4f2 and Isl1 in RGC fate commitment, because the RGCs, although abnormal, still can form, migrate to the inner side of the retina, and project axons in the absence of Isl1 and/or Pou4f2 (23,25,28). Interestingly, RGCs in Pou4f2-knockout mice assume a hybrid identity, expressing a mixture of marker genes for RGCs, amacrine cells, and horizontal cells, suggesting that Pou4f2 indeed may play a role as an RGC specifier (33,34). Based on these considerations, we hypothesized that the RGC fate is determined by a core group of transcription factors and that Pou4f2 and Isl1 belong to this core group.…”