Eomesodermin, a target gene of Pou4f2, is required for retinal ganglion cell and optic nerve development in the mouse.

The mechanisms regulating retinal ganglion cell (RGC) development are crucial for retinogenesis and for the establishment of normal vision. However, these mechanisms are only vaguely understood. RGCs are the first neuronal lineage to segregate from pluripotent progenitors in the developing retina. As output neurons, RGCs display developmental features very distinct from those of the other retinal cell types. To better understand RGC development, we have previously constructed a gene regulatory network featuring a hierarchical cascade of transcription factors that ultimately controls the expression of downstream effector genes. This has revealed the existence of a Pou domain transcription factor, Pou4f2, that occupies a key node in the RGC gene regulatory network and that is essential for RGC differentiation. However, little is known about the genes that connect upstream regulatory genes, such as Pou4f2 with downstream effector genes responsible for RGC differentiation. The purpose of this study was to characterize the retinal function of eomesodermin (Eomes), a T-box transcription factor with previously unsuspected roles in retinogenesis. We show that Eomes is expressed in developing RGCs and is a mediator of Pou4f2 function. Pou4f2 directly regulates Eomes expression through a cis-regulatory element within a conserved retinal enhancer. Deleting Eomes in the developing retina causes defects reminiscent of those in Pou4f2(-/-) retinas. Moreover, myelin ensheathment in the optic nerves of Eomes(-/-) embryos is severely impaired, suggesting that Eomes regulates this process. We conclude that Eomes is a crucial regulator positioned immediately downstream of Pou4f2 and is required for RGC differentiation and optic nerve development

Birth of cone bipolar cells, but not rod bipolar cells, is associated with existing RGCs.

Retinal ganglion cells (RGCs) play important roles in retinogenesis. They are required for normal retinal histogenesis and retinal cell number balance. Developmental RGC loss is typically characterized by initial retinal neuronal number imbalance and subsequent loss of retinal neurons. However, it is not clear whether loss of a specific non-RGC cell type in the RGC-depleted retina is due to reduced cell production or subsequent degeneration. Taking advantage of three knockout mice with varying degrees of RGC depletion, we re-examined bipolar cell production in these retinas from various aspects. Results show that generation of the cone bipolar cells is correlated with the existing number of RGCs. However, generation of the rod bipolar cells is unaffected by RGC shortage. Results report the first observation that RGCs selectively influence the genesis of subsequent retinal cell types

Differential Susceptibility of Retinal Neurons to the Loss of Mitochondrial Biogenesis Factor Nrf1

The retina, the accessible part of the central nervous system, has served as a model system to study the relationship between energy utilization and metabolite supply. When the metabolite supply cannot match the energy demand, retinal neurons are at risk of death. As the powerhouse of eukaryotic cells, mitochondria play a pivotal role in generating ATP, produce precursors for macromolecules, maintain the redox homeostasis, and function as waste management centers for various types of metabolic intermediates. Mitochondrial dysfunction has been implicated in the pathologies of a number of degenerative retinal diseases. It is well known that photoreceptors are particularly vulnerable to mutations affecting mitochondrial function due to their high energy demand and susceptibility to oxidative stress. However, it is unclear how defective mitochondria affect other retinal neurons. Nuclear respiratory factor 1 (Nrf1) is the major transcriptional regulator of mitochondrial biogenesis, and loss of Nrf1 leads to defective mitochondria biogenesis and eventually cell death. Here, we investigated how different retinal neurons respond to the loss of Nrf1. We provide in vivo evidence that the disruption of Nrf1-mediated mitochondrial biogenesis results in a slow, progressive degeneration of all retinal cell types examined, although they present different sensitivity to the deletion of Nrf1, which implicates differential energy demand and utilization, as well as tolerance to mitochondria defects in different neuronal cells. Furthermore, transcriptome analysis on rod-specific Nrf1 deletion uncovered a previously unknown role of Nrf1 in maintaining genome stability

Creation of cis-regulatory elements during sea urchin evolution by co-option and optimization of a repetitive sequence adjacent to the spec2a gene

AbstractThe creation, preservation, and degeneration of cis-regulatory elements controlling developmental gene expression are fundamental genome-level evolutionary processes about which little is known. Here, we identify critical differences in cis-regulatory elements controlling the expression of the sea urchin aboral ectoderm-specific spec genes. We found multiple copies of a repetitive sequence element termed RSR in genomes of species within the Strongylocentrotidae family, but RSRs were not detected in genomes of species outside Strongylocentrotidae. spec genes in Strongylocentrotus purpuratus are invariably associated with RSRs, and the spec2a RSR functioned as a transcriptional enhancer and displayed greater activity than did spec1 or spec2c RSRs. Single-base pair differences at two cis-regulatory elements within the spec2a RSR increased the binding affinities of four transcription factors, SpCCAAT-binding factor at one element and SpOtx, SpGoosecoid, and SpGATA-E at another. The cis-regulatory elements to which these four factors bound were recent evolutionary acquisitions that acted to either activate or repress transcription, depending on the cell type. These elements were found in the spec2a RSR ortholog in Strongylocentrotus pallidus but not in RSR orthologs of Strongylocentrotus droebachiensis or Hemicentrotus pulcherrimus. Our results indicated that a dynamic pattern of cis-regulatory element evolution exists for spec genes despite their conserved aboral ectoderm expression

Phospho-Histone-3 and BrdU labeling show unexpected proliferation dynamics in the central retinas that have varying degrees of RGC loss.

<p>A–H. Representative confocal images at P0 and P4 show PH3-positive cells (green) in cryosections of wildtype and RGC-depleted retinas. I–L. Representative confocal images at P4 show PH3-positive cells in flat-mounted retinas. M–T. Representative confocal images at P0 and P4 show BrdU-positive cells (green) in cryosections. Red: PI. All scale bars  = 50 µm. U–W. Histograms comparing the numbers of proliferating cells estimated by PH3-positive cells in sections (U), PH3-positive cells in flat-mounts (V), and BrdU-positive cells in sections (W). No proliferating cells were found in the sampled region in all examined retinas at P7 and P10.</p

Birthdating experiments confirm fewer C-BPC and normal R-BPC production in the <i>Atoh7^−/−</i> retina.

<p>A, B. Representative confocal images of retinal cryosections from mice that received BrdU injection at P1 and harvested at P20. In the inner nuclear layer, Chx10 antibody (red) labels both R-BPCs and C-BPCs; PKCα antibody (green) labels only R-BPCs; and BrdU (blue) marks the cells born at P1. A′ and B′ show only the BrdU channel at a positions corresponding to A and B. Arrows indicate R-BPCs and arrowheads indicate C-BPCs. Scale bar  = 50 µm. Cells outside of the inner nuclear layer were not analyzed. Images of retinas with BrdU injected at other time points are not shown. C, D. Statistical analysis comparing C-BPC (C) and R-BPC (D) cell counts that were born on a specific day. Significantly fewer C-BPCs were born in the <i>Atoh7^−/−</i> retina on each examined day. In the sampling area, no C-BPCs were observed in either wildtype or <i>Atoh7^−/−</i> retinas born at P7.</p

Assessment of C-BPC and R-BPC numbers show reduced C-BPC numbers and normal R-BPC numbers in the RGC-depleted retinas.

<p>A–P. Representative confocal images from the central retinas from wildtype, <i>Pou4f2^−/−, Atoh7^−/−</i>, and DKO mice at P5, P10, P15, and P21. Samples were labeled with Chx10- (red) and PKCα- (green) to depict total BPCs and R-BPCs respectively. Nuclei were counterstained with TOPRO-3 (blue). Scale bar  = 50 µm. Q–S. Histograms comparing the mean numbers of total BPCs (Q), R-BPCs (R), and C-BPCs (S) in four different retinas with varying degrees of RGC depletion. Means of the C-BPC populations were calculated by taking the difference between Chx10- and PKCα- positive cell counts.</p

Assessment of apoptotic BPCs show a higher rate of apoptotic BPCs in the wildtype retina.

<p>A–L. Representative confocal images of the central retinas from wildtype, <i>Pou4f2^−/−, Atoh7^−/−</i>, and DKO mice at P5, P10 and P15. Retinas were labeled with antibodies to Chx10 (red), cleaved Caspase-3 (green) and TOPRO-3 (blue). Scale bar  = 50 µm. M. A histogram showing the percentage of Caspase-3 and Chx10-double positive cells over the Chx10-positive cells, depicting the ratio of apoptotic bipolar cells over all bipolar cells in four different retinas.</p

The number of <i>Vsx1</i>-expressing cells supports that fewer C-BPCs are found in the <i>Atoh7^−/−</i> retina.

<p>A–F. Confocal images of cryosections showing <i>Vsx1^LacZ/+</i>-expressing C-BPCs (green) in the wildtype (<i>Atoh7^+/−;Vsx1^LacZ/+</i>) and <i>Atoh7^−/−</i> (<i>Atoh7^−/−;Vsx1^LacZ/+</i>) retinas. Green: LacZ; red: propidium iodide. Scale bar  = 50 µm. G. A histogram comparing the mean number of <i>Vsx1^LacZ</i>-positive cells in wildtype and <i>Atoh7^−/−</i> retinas.</p

Phospho-Histone-3 and BrdU labeling of the peripheral retinas show the number of proliferating cells corresponds to the existing RGC number.

<p>A–H. Representative confocal images at P0 and P4 show PH3-positive cells (green) in cryosections of wildtype and RGC-depleted retinas. I–L. Representative confocal images at P4 show PH3-positive cells in flat-mounted retinas. Dash lines mark the edge of the retina. M–T. Representative confocal images at P4 and P7 show BrdU-positive cells (green) in cryosections. Red in all panels: propidium iodide. All scale bars  = 50 µm. U–W. Histograms comparing the numbers of proliferating cells estimated by PH3-positive cells in sections (U), PH3-positive cells in flat-mounts (V), and BrdU-positive cells in sections (W). Proliferating cell numbers found in all retinas at P10 are either zero or close to zero. There is no significant difference between any two genotypes at P10.</p