Abstract
Retinal ganglion cells (RGCs) provide the only output of the retina, with their axons projecting to central nervous system targets. The combinatorial roles of homeodomain (HD) and basic helix-loop-helix (bHLH) transcription factors (TFs) determine RGC differentiation. The Class IV POU-domain proteins, BRN3a, BRN3b, and BRN3c, are all expressed in RGC. However, only Brn3b deletion leads to a major defect in RGC differentiation and axonal guidance. Dlx1/Dlx2 double knockout mice have 33% loss of late-born RGCs. Vax2 is restricted to ventral RGC and maintains ventral RGC axonal projections to their target, the medial rostral superior colliculus (SC). Isl1 defines a distinct but overlapping subpopulation of RGCs with Brn3b, whereas Isl2 specifies the contralateral projection of RGC axons.
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1 Introduction
Among the six major classes of retinal neurons, retinal ganglion cells (RGC) are the only projection neurons. RGCs differentiate in the mouse retina around E11.5 followed by horizontal, cone, and amacrine cells. The early generated RGC are located in the central inner retina, and the axons of these RGC commence their journey to central nervous system (CNS) targets immediately after terminal differentiation (Marquardt and Gruss 2002). Combinations of homeodomain (HD) and basic helix-loop-helix (bHLH) transcription factors (TF) play important roles in RGC cell fate specification and influence RGC axonal pathfinding choices en route. In this review, we will focus on the roles of HD transcription factors in these processes.
2 Brn-3 Genes
The POU-domain is a bipartite DNA-binding protein domain, containing a POU-specific region and a POU-homeodomain region. The class IV POU-domain proteins, BRN3a, BRN3b, and BRN3c (POU4f1, POU4f2, and POU4f3, respectively) are the homologs of Unc-86 in C. elegans. Brn-3 genes are expressed in the embryonic and adult CNS, and are required for sensorineural development and survival.
All three Brn-3 POU-homeodomain genes are expressed in the developing retina, specifically in postmitotic RGC. Brn3b expression is first detected in the earliest RGC at E11.5 in the central inner mouse retina, followed by Brn3a and Brn3c expression 2 days later (Xiang et al. 1995; Pan et al. 2005). Through E15.5 to the adult retina, Brn3a and Brn3b expression overlaps in 80% of RGC. However, Brn3a is the predominant gene expressed in P5 retinas, with few RGC expressing Brn3b (Quina et al. 2005). Only ∼15% of RGC express Brn3c (Xiang et al. 1995).
The overlapping expression pattern and a similar specific DNA-binding site [(A/G)CTCATTAA(T/C)] of these three BRN-3 proteins suggest their functional redundancy in retinogenesis. However, targeted mutations of Brn-3 genes in mice show distinct defects, but only the Brn3b −/− mouse shows an obvious retinal phenotype. In Brn3b null mice there is loss of 60–80% RGC in adult retinas, depending on the background genetic strain (Erkman et al. 1996; Gan et al. 1996). This RGC loss is due to enhanced apoptosis after E15.5, but not to defects of initial cell fate specification or migration. Brn3b is also required for RGC axon pathfinding and fasciculation (Erkman et al. 2000). Brn3a null mutants die at birth, with loss of dorsal root ganglion and trigeminal neurons (Erkman et al. 1996; Xiang et al. 1996). Brn3c −/− mice display deficits in balance and complete deafness, attributed to loss of vestibular and auditory hair cells (Erkman et al. 1996; Xiang et al. 1997). Neither Brn3a nor Brn3c mutants show obvious defects in retinal development.
Brn3a and Brn3c are identified as downstream of Brn3b in retinogenesis, and there is reduced Brn3a expression in Brn3b mutants (Erkman et al. 1996). Despite the dominant roles of Brn3b in retinal development, several independent groups have reported that all three Brn-3 genes are functionally equivalent in retinogenesis. Overexpression of Brn3a, Brn3b, or Brn3c in chick retinal progenitors exerts a similar effect in promoting RGC differentiation (Liu et al. 2000). Knocking-in the Brn3a coding sequence into a Brn3b null background mouse rescues RGC from apoptosis and restores RGC axonal pathfinding (Pan et al. 2005).
A recent study has shown that conditional deletion of Brn3a alters RGC dendritic stratification without influencing RGC axon central projections. However, the conditional Brn3b knockout mice show reduced RGC numbers, loss of axonal projections to medial (MTN) and lateral terminal nuclei (LTN) and corresponding visual sensory defects (Badea et al. 2009).
3 Dlx Genes
Dlx genes are the vertebrate orthologs of distal-less (Dll). There are six Dlx genes identified in mice, which are arranged into three bigene clusters (Dlx1/Dlx2, Dlx5/Dlx6, and Dlx3/Dlx7), and are localized on mouse chromosomes 2, 6, and 11, respectively (Ghanem et al. 2003). Within the intergenic regions of Dlx1/2 and Dlx5/6, several cis-acting regulators have been characterized, including I12a and I12b between the Dlx1 and Dlx2 genes, and I56i and I56ii separating Dlx5 and Dlx6 (Poitras et al. 2007). Two conserved enhancer elements, URE1 and URE2, have also been found in the 5′ flanking region of Dlx1 (Hamilton et al. 2005) (Du and Eisenstat, unpublished). These cis-acting elements are important for cross-regulatory interactions between the Dlx genes. One example is that DLX1 and DLX2 regulate Dlx5/Dlx6 expression by acting on I56i (Zhou et al. 2004).
Dlx1 and Dlx2 were first detected in the retinal neuroepithelium on E12.5 including mitotic cells adjacent to the ophthalmic ventricle (Eisenstat et al. 1999). Our recent study reported DLX2 immunostaining in E11.5 retina, with DLX2 expressed in a dorsal (high) to ventral (low) gradient (de Melo et al. 2008). At E13.5, both DLX1 and DLX2 are expressed throughout the retina, with boundaries in peripheral and central inner retina. Interestingly, some other “retinal” homeobox genes are expressed in a nearly complementary manner to DLX2 at this stage. At E13.5, the highest level of PAX6 expression is observed in the most peripheral retina and BRN3b is expressed in the inner central retina, where DLX2 is absent. By E18.5, DLX1 and DLX2 expressions are highly restricted to the ganglion cell layer (GCL) and inner part of the neuroblastic layer (NBL), where they are co-expressed with markers for RGC, amacrine, and horizontal cells. DLX1 expression resembles DLX2 in embryonic retina, but decreases dramatically after birth and cannot be detected in adult retina. However, DLX2 is robustly expressed in the GCL and inner nuclear layer (INL) throughout adulthood (de Melo et al. 2003).
Although the role of Dlx genes in forebrain development is well reported, very few studies have described Dlx gene function in retina development. Homozygous deletion of Dlx1 and Dlx2 is perinatally lethal, and leads to a 33% reduction of RGC number due to enhanced apoptosis of late-born RGCs (de Melo et al. 2005). TrkB, a receptor for brain derived neurotrophic factor (BDNF) mediated signalling, was identified as a DLX2 downstream target during mouse retinal development and may contribute to RGC survival (de Melo et al. 2008).
The role of Dlx5 and Dlx6 genes in retinogenesis is still not clear. In situ hybridization revealed Dlx5 mRNA expression in retina by E16.5. In P0 and adult retina, Dlx5 mRNA is co-expressed with DLX2 in the GCL and INL. The Dlx5/Dlx6 intergenic enhancer (I56i) is co-expressed with DLX5, DLX1, and DLX2 in RGC, amacrine, and horizontal cells (Zhou et al. 2004). There is no published report regarding the retinal phenotype of Dlx5/Dlx6 knockout mice.
4 Vax Genes
Vax (Ventral anterior homeobox-containing) genes are a homeodomain gene subfamily, closely related to Emx genes, sharing sequence homology, similar chromosomal location, and expression patterns (Hallonet et al. 1998). In the mouse, Vax1 mRNA is first detected at E8, in the anterior neural ridge and adjacent ectoderm. During embryogenesis, Vax1 expression is restricted to the derivatives of these regions, including basal forebrain, ventral optic vesicle, optic disk, stalk, and chiasm (Hallonet et al. 1998). The targeted deletion of Vax1 shows defects in RGC axonogenesis and axonal-glial associations, without influencing expression of Pax2 and BF1. In addition, Vax1 −/− axons fail to fasciculate and do not extend toward the hypothalamic midline, leading to an absence of optic chiasm development. These RGC axon pathfinding defects are partially due to the loss of some important axon guidance cues, including Netrin-1 and EphB3, but not Slit1(Bertuzzi et al. 1999). Another obvious phenotype of Vax1 mutants is the failure of choroid fissure closure, known as coloboma. Pax6 and Rx are ectopically expressed in the Vax1 mutant optic nerve. However, Pax2 expression remains unaffected in the mutants.
VAX2 shares an identical homeodomain with VAX1, and the Vax2 gene is tightly linked with Emx1 in mouse and human. By E9, Vax2 transcripts are detected in the ventral optic vesicle, with lower expression in the optic nerve and stalk. Vax1 and Vax2 then share overlapping expression patterns in ventral retina and optic stalk. By E12, Vax2 expression is restricted to the ventral neural retina in the whole retinal population. However, at later embryonic stages, Vax2 is only detected in ventral RGC. Vax2 is not expressed in the adult retina (Bertuzzi et al. 1999; Mui et al. 2002).
Consistent with its predominant ventral retinal expression pattern, Vax2 plays a major role in ventralizing embryonic retina. Misexpression of Vax2 in the dorsal retina is able to alter the expression of the putative dorsal-ventral marker genes, including upregulation of ventral retinal markers EphB2/EphB3, Pax2, and Vax2 itself, and downregulation of the dorsally restricted TF, Tbx5 (Barbieri et al. 1999). In addition, ectopic Vax2 expression in dorsal retina is sufficient to induce profound axon pathfinding defects of dorsal RGC (Schulte et al. 1999). In agreement with these Vax2 gain-of-function studies, the ventral RGC from Vax2 null mice show complete dorsalization. The RGC axons from Vax2 −/− ventral retina aberrantly project to the lateral rostral edge of the superior colliculus (SC) together with all the dorsal RGC axons, instead of medial rostral SC, the destination of all the wild-type ventral RGC axons. The expression of EphB2/EphB3 is absent in Vax2 −/− ventral retina.
5 Islet Genes
ISL1 and ISL2 are a subfamily of LIM homeodomain TF, characterized by two zinc-finger motifs (LIM domain) and a homeodomain. Both ISL1 and ISL2 play important roles in determining motor neuron subtype identity, axonal projections and peripheral innervations (Shirasaki and Pfaff 2002). Most of the work has been done in the spinal cord of vertebrate and invertebrate models. Isl1 and Isl2 are also expressed in the embryonic and postnatal retina.
Isl2 expression is first detected in the E13 retina, and by E17, almost all the Isl2 positive cells are RGC (Pak et al. 2004). Isl2 is expressed at high levels in the dorsal retina, with weak expression in the ventral-temporal region. By repressing Zic2 and EphB1, Isl2 specifies the contralateral projection of RGC axons. In comparison to the specific RGC expression of Isl2, Isl1 expression shows different patterns from embryonic to postnatal retina. Prior to E15.5, Isl1 is predominantly expressed in RGC. However, from E15.5 to the adulthood, Isl1 expression is detected in RGC, amacrine cells, and bipolar cells (Elshatory et al. 2007). Recent work has shown that under the regulation of ATOH7 (formerly MATH5), Isl1 defines a distinct but overlapping subpopulation of RGC with Brn3b (Mu et al. 2008; Pan et al. 2008)
6 Summary
In this review, we have described four major families of homeobox genes which play important roles in RGC differentiation as well as axonal pathfinding. The mechanism underlying how these HD TFs affect axonal pathfinding is not entirely known. One possibility is that the downstream targets directly regulated by these HD TF are responsible for axonal guidance. Examples of this are the repression of EphB1 by Isl2, and Vax1/Vax2 regulation of EphB2/EphB3 expression. The roles of Dlx homeobox genes in RGC axonal guidance have not yet been reported. However, in the mouse telencephalon, Dlx1 and Dlx2 promote the tangential migration of GABAergic interneurons by repressing axonal growth (Cobos et al. 2007) and inhibiting Neuropilin-2 expression(Le et al. 2007). It is possible that the genetic program defining RGC identity also encodes a unique “sensory” network for their axons, determining how and where RGC axons respond to guidance cues en route to CNS targets.
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Zhang, Q., Eisenstat, D.D. (2012). Roles of Homeobox Genes in Retinal Ganglion Cell Differentiation and Axonal Guidance. In: LaVail, M., Ash, J., Anderson, R., Hollyfield, J., Grimm, C. (eds) Retinal Degenerative Diseases. Advances in Experimental Medicine and Biology, vol 723. Springer, Boston, MA. https://doi.org/10.1007/978-1-4614-0631-0_87
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