Mapping membrane lipids in the developing and adult mouse retina under physiological and pathological conditions using mass spectrometry.

Membrane phospholipids play pivotal roles in various cellular processes, and their levels are tightly regulated. In the retina, phospholipids had been scrutinized because of their distinct composition and requirement in visual transduction. However, how lipid composition changes during retinal development remains unclear. Here, we used liquid chromatography-mass spectrometry (LC-MS) to assess the dynamic changes in the levels of two main glycerophospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), in the developing mouse retina under physiological and pathological conditions. The total levels of PC and PE increased during retinal development, and individual lipid species exhibited distinct level changes. The amount of very-long-chain PC and PE increased dramatically in the late stages of retinal development. The mRNA levels of Elovl2 and Elovl4, genes encoding enzymes essential for the synthesis of very-long-chain polyunsaturated fatty acids, increased in developing photoreceptors. Cell sorting based on CD73 expression followed by LC-MS revealed distinct changes in PC and PE levels in CD73-positive rod photoreceptors and CD73-negative retinal cells. Finally, using the NaIO3-induced photoreceptor degeneration model, we identified photoreceptor-specific changes in PC and PE levels from 1 day after NaIO3 administration, before the outer segment of photoreceptors displayed morphological impairment. In conclusion, our findings provide insight into the dynamic changes in PC and PE levels in the developing and adult mouse retina under physiological and pathological conditions. Furthermore, we provide evidence that cell sorting followed by LC-MS is a promising approach for investigating the relevance of lipid homeostasis in the function of different retinal cell types.

The role of Zhx2 transcription factor in bipolar cell differentiation during mouse retinal development.

We found that the Zhx2 gene (whose product is known to act as a tumor suppressor in hepatocellular carcinoma) is expressed in embryonic retinal progenitors and in developing cone bipolar cells in the postnatal retina, as well as in Müller glia in the mature retina. To examine the functions of Zhx2 protein during retinal development, we performed loss- and gain-of-function analyses using a retinal explant culture system. We introduced a plasmid encoding Zhx2 shRNA into isolated mouse retinas at E17.5, and the retinas were cultured as explants. After 3 days of culture, proliferation was slightly enhanced, leading to retinas thicker than in the control, but this phenomenon was observed only transiently. After 14 days of the culture, the thickness and gross morphology of retinas expressing sh-Zhx2 were indistinguishable from those of the control. The numbers of rod cells, amacrine cells, and Müller glia were the same in both groups. However, although the total number of bipolar cells was the same, the experimental group saw an increased population of ON bipolar cells, and decreased numbers of a subset of OFF bipolar cells. We also examined the effects of overexpression of Zhx2. Although Zhx2 acts as a tumor suppressor, its overexpression in developing retinas did not lead to any discernible difference in retinal thickness, suggesting that proliferation activity was not affected. After 14 days of explant culture, the total number of bipolar cells decreased, and subset composition was altered. Taken together, these results suggest that Zhx2 plays roles in the regulation of bipolar cell subset fate determination during retinal development.

Histone demethylase Jmjd3 is required for the development of subsets of retinal bipolar cells.

Di- and trimethylation of lysine 27 on histone H3 (H3K27me2/3) is an important gene repression mechanism. H3K27me2/3-specific demethylase, Jmjd3, was expressed in the inner nuclear layer during late retinal development. In contrast, H3K27 methyltransferase, Ezh2, was highly expressed in the embryonic retina but its expression decreased rapidly after birth. Jmjd3 loss of function in the developing retina resulted in failed differentiation of PKC-positive bipolar cell subsets (rod-ON-BP) and reduced transcription factor Bhlhb4 expression, which is critical for the differentiation of rod-ON-BP cells. Overexpression of Bhlhb4, but not of other BP cell-related genes, such as transcription factors Neurod and Chx10, in Jmjd3-knockdown retina rescued loss of PKC-positive BP cells. Populations of other retinal cell subsets were not significantly affected. In addition, proliferation activity and apoptotic cell number during retinal development were not affected by the loss of Jmjd3. Levels of histone H3 trimethyl Lys27 (H3K27me3) in the Bhlhb4 locus were lower in Islet-1-positive BP cells and amacrine cells than in the Islet-1-negative cell fraction. The Islet-1-negative cell fraction consisted mainly of photoreceptors, suggestive of lineage-specific demethylation of H3K27me3 in the Bhlhb4 locus. We propose that lineage-specific H3K27me3 demethylation of critical gene loci by spatiotemporal-specific Jmjd3 expression is required for appropriate maturation of retinal cells.

Role of transcription factor Tgif2 in photoreceptor differentiation in the mouse retina.

5'TG3'-interacting factors (TGIFs) function as transcriptional repressors. Defects in TGIFs cause severe abnormalities in the developing brain and face. We found that Tgif2 was highly expressed in the mouse retina at early stages of development and examined its role in retinal development. Knockdown of Tgif2 in retinal explants at E14 using shRNA (sh-Tgif2) resulted in a decreased number of rod photoreceptors, whereas the number of cone photoreceptors increased without perturbation of cell proliferation and apoptosis. Concomitantly, the expression levels of photoreceptor-related genes were decreased in sh-Tgif2-introduced retinal explants. To examine the in vivo effects of Tgif2 overexpression, we generated Tgif2 conditional knock-in mice (Tgif2cKI). Although retinal cell differentiation, based on the relative proportions of retinal subtypes in the mature retina, was not affected, we observed abnormal localization of cone photoreceptor cell nuclei in the outer nuclear layer of the Tgif2cKI retina. However, electrical retinography suggest that cones in Tgif2cKI were functionally equivalent to those of wild mice. Our study revealed that Tgif2 participates in photoreceptor cell differentiation in the early stages of retinal development and regulates proper subretinal localization of the cone photoreceptors.

Ablation of Kcnj10 expression in retinal explants revealed pivotal roles for Kcnj10 in the proliferation and development of Müller glia.

PURPOSE: We previously found that Kcnj10, an inwardly-rectifying potassium channel, is a gene expressed in c-kit-positive retinal progenitor cells on P1. The shRNA-mediated screening of the functions of the genes for retinal development in retinal explant culture suggested a role for Kcnj10 in the differentiation of 23Müller glia. In the present study, we extended the work and focused on analyzing the role of Kcnj10 in retinal development. METHODS: shRNA-mediated downregulation of Kcnj10 in retinal explants and the in vivo mouse retina at the P1 stage was performed. Differentiation and proliferation of the retina were examined with immunohistochemistry. The effect of barium (Ba(2+)) treatment, which inhibits potassium currents by blocking potassium channels, on retinal development was examined. RESULTS: When Kcnj10 was downregulated at E18, cellular proliferation and morphological differentiation were perturbed; in particular, a decreased number of Müller glial cells with abnormal morphological maturation was observed. The overexpression of Kcnj10 in retinal progenitors did not result in gross abnormality during retinal development, but rescued the abnormal differentiation induced with sh-Kcnj10. The presence of Ba(2+) in the retinal explant medium led to a phenotype similar to that seen with sh-Kcnj10. Ba(2+) exerts an effect mainly during late retinal development, and sh-Kcnj10 in the P1 retina affected Müller glia maturation, suggesting that Kcnj10 plays a pivotal role in the maturation of retinal cell subsets. A previous study of Kcnj10-knockout mice showed no obvious abnormality in retinal differentiation, especially of Müller glia. We examined the effects of the downregulation of Kcnj10 with in vivo electroporation of sh-Kcnj10 in the P1 retina. Retinal differentiation was perturbed, as seen following the in vitro downregulation of Kcnj10, suggesting that compensatory gene expression and/or signaling occurred in the Kcnj10-knockout mice in the retina, leading to normal eye development. CONCLUSION: Kcnj10 plays a role in Müller glia maturation during retinal development probably through ionic channel activities.

Setd5, but not Setd2, is indispensable for retinal cell survival and proliferation.

Trimethylation of histone H3 at lysine 36 (H3K36me3) is associated with active transcription. We used mouse retinal explant cultures and shRNA to investigate the roles of Setd2 and Setd5, which encode H3K36me3 methyltransferases, in retinal development. We found that shSetd5 caused abnormal retinal structures and reduced rods and Müller cells, whereas shSetd2 did not cause any abnormalities. The mutant SETD5 lacking the SET domain failed to reverse the phenotypes observed in the shSetd5-expressing retinas, while SETD5S1257*, which does not interact with HDAC3 and PAF1 complexes, rescued proliferation, but not apoptosis, induced by shSetd5. Taken together, we found that Setd5, but not Setd2, is essential for sustaining retinal cell survival and proliferation, and the SET domain of SETD5 is pivotal for both functions.

Loss-of-function approach using mouse retinal explants showed pivotal roles of Nmnat2 in early and middle stages of retinal development.

Nicotinamide mononucleotide adenylyltransferase (Nmnat) is a class of enzymes with three members (Nmnat1-3). Nmnat1 is in nucleus and associated with Leber congenital amaurosis, a form of early-onset retinal degeneration, while Nmnat2 is in cytoplasm and a well-characterized neuroprotective factor. The differences in their biological roles in the retina are unclear. We performed short hairpin RNA (shRNA)-based loss-of-function analysis of Nmnat2 during mouse retinal development in retinal explant cultures prepared from early (E14.5), middle (E17.5), or late (postnatal day [P]0.5) developmental stages. Nmnat2 has important roles in the survival of retinal cells in the early and middle stages of retinal development. Retinal cell death caused by Nmnat2 knockdown could be partially rescued by supplementation with NAD or nicotinamide mononucleotide (NMN). Survival of retinal cells in the late stage of retinal development was unaffected by Nmnat2, but differentiation of Müller glia was controlled by Nmnat2. RNA-Seq analyses showed perturbation of gene expression patterns by shRNAs specific for Nmnat1 or Nmnat2, but gene ontology analysis did not provide a rational explanation for the phenotype. This study showed that Nmnat2 has multiple developmental stage-dependent roles during mouse retinal development, which were clearly different from those of Nmnat1, suggesting specific roles for Nmnat1 and Nmnat2.

Involvement of metabotropic glutamate receptor 5 signaling in activity-related proliferation of adult hippocampal neural stem cells.

Adult hippocampal neural stem cells can be activated by hippocampal neural activities. When focal cerebral ischemia, known as middle cerebral artery occlusion (MCAO), occurs, neural stem cells are activated to promote their proliferation. However, the mechanism by which these cells are activated is still unclear. Here, we indicate the involvement of metabotropic glutamate receptor 5 (mGluR5) signaling in neural stem cells in their activity-related proliferation after MCAO. We found mGluR5 molecules on neural stem cells by using calcium imaging. We detected the activation of neural stem cells by adding the mGluR5 agonist (RS)-2-chloro-5-hydroxyphenylglycine. On a hippocampal slice, the activation of neural stem cells to promote their proliferation was initiated by theta-burst electrical stimulation at the perforant pathway, and this activation was significantly blocked by an mGluR5 antagonist, 2-methyl-6-(phenylethynyl)pyridine (MPEP). In addition to this, the injection of the blood-brain barrier-permeable mGluR5 agonist 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide into live mice promoted the proliferation of neural stem cells. Moreover, in vivo theta-burst electrical stimulation induced proliferation of neural stem cells. A chronic field recording study showed that the activity of the hippocampal formation was elevated after MCAO. Finally, we observed that the mGluR5 antagonist MPEP significantly blocked the stimulated proliferation of neural stem cells induced by MCAO, by blocking mGluR5 signaling. Our results suggest that glutamates released by the elevated neural activities after MCAO may trigger mGluR5 signaling in neural stem cells to promote their proliferation.

Cholinergic activation of hippocampal neural stem cells in aged dentate gyrus.

Adult hippocampal neurogenesis contributes to the hippocampal circuit's role in cognitive functioning. New neurons are generated from hippocampal neural stem cells (NSCs) throughout life, but their generation is substantially diminished in aged animals due to a decrease in NSC proliferation. Because acetylcholine (ACh) is an important neurotransmitter released in the hippocampus during learning and exercise that is known to decrease with aging, we investigated whether aged NSCs can respond to ACh. In this study, we found that cholinergic stimulation has a positive effect on NSC proliferation in both young adult (8-12 weeks old) and aged mice (>2 years old). In fresh hippocampal slices, we observed a rapid calcium increase in NSCs in the dentate gyrus after muscarinic cholinergic stimulation, in both age groups. Furthermore, we found that the exercise-induced promotion of aged NSC proliferation was abrogated by the specific lesioning of the septal cholinergic system. In turn, cholinergic activation by either eserine (physostigmine) or donepezil treatment promoted the proliferation of NSCs in aged mice. These results indicate that NSCs respond to cholinergic stimulation by proliferating in aged animals. Physiological and/or pharmacological cholinergic stimulation(s) may ameliorate cognitive decline in aged animals, by supporting adult hippocampal neurogenesis.

Identification of CD44 as a cell surface marker for Müller glia precursor cells.

In the retina, both neurons and glia differentiate from a common progenitor population. CD44 cell surface antigen is a hyaluronic acid receptor expressed on mature Müller glial cells. We found that in the developing mouse retina, expression of CD44 was transiently observed at or around birth in a subpopulation of c-kit-positive retinal progenitor cells. During in vitro culture, purified CD44/c-kit-positive retinal progenitor cells exclusively differentiated into Müller glial cells and not into neurons, suggesting that CD44 marks a subpopulation of retinal progenitor cells that are fated to become glia. Over-expression of CD44 inhibited the extension of processes by Müller glial cells and neurons. Notch signaling is known to be involved in the specification of retinal progenitors into a glial fate. Activation of Notch signaling increased the number of CD44-positive cells, and treatment with the Notch signal inhibitor, DAPT, at early, but not later, stages of retinal development abolished both CD44-positive cells and Müller glial cells. Together, CD44 was identified as an early cell surface marker of the Müller glia lineage, and Notch signalling was involved in commitment of retinal progenitor cells to CD44 positive Müller glial precursor cells.

Roles of Nmnat1 in the survival of retinal progenitors through the regulation of pro-apoptotic gene expression via histone acetylation.

Leber congenital amaurosis (LCA) is a severe, genetically heterogeneous dystrophy of the retina and mutations in the nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) gene is one of causal factors of LCA. NMNAT1 is a nuclear enzyme essential for nicotinamide adenine dinucleotide (NAD) biosynthesis pathways, but the mechanisms underlying the LCA pathology and whether NMNAT1 has a role in normal retinal development remain unclear. Thus, we examined the roles of Nmnat1 in retinal development via short hairpin (sh)-RNA-mediated downregulation. Retinal explants expressing sh-Nmnat1 showed large numbers of apoptotic retinal progenitor cells in the inner half of the neuroblastic layer. Decreased intracellular NAD content was observed and the addition of NAD to the culture medium attenuated sh-Nmnat1-induced apoptosis. Of the nuclear Sirtuin (Sirt) family, the expression of sh-Sirt1 and sh-Sirt6 resulted in a phenotype similar to that of sh-Nmnat1. Sirt proteins are histone deacetylases and the expression of sh-Nmnat1 increased the levels of acetylated histones H3 and H4 in the retina. Expression of sh-Nmnat1 resulted in significantly increased expression of Noxa and Fas, two pro-apoptotic genes. Acetylation of the genomic 5'-untranslated regions of Noxa and Fas loci was upregulated by sh-Nmnat1 expression. The co-expression of sh-Fas with sh-Nmnat1 reduced the number of apoptotic cells induced by sh-Nmnat1 expression alone. Taken together, our data suggested that the increased expression of Noxa and Fas explains, at least in part, the phenotype associated with sh-Nmnat1 in the retina. Taken together, these findings demonstrate the importance of the NAD biosynthesis pathway in normal development of the retina.

Forkhead Box Protein P1 Is Dispensable for Retina but Essential for Lens Development.

Purpose: Forkhead box protein P1 (Foxp1) is a transcriptional repressor expressed in many tissues. We identified Foxp1 as a highly expressed gene in retinal progenitor cells and investigated its roles during eye development. Methods: Mouse eyes with Foxp1 gain- or loss-of-function were established in vitro and in vivo. Results: Foxp1 overexpression in retinal progenitor cells resulted in reduced rod and increased cone photoreceptors. However, retina-specific knockout of Foxp1 was not associated with retinal differentiation abnormalities. Foxp1 was highly expressed in the lens during early development, and continued to be expressed in epithelial and cortical fiber cells until adulthood. At birth, analyses of Foxp1 lens-specific knockout (Foxp1-L-CKO) mice showed no gross morphologic changes in germinal or central epithelial cell compared to the controls. However, the numbers of proliferating and apoptotic cells were significantly increased in Foxp1-L-CKO mice. In addition, clear Y-structures were not observed in either the posterior or anterior sutures of the Foxp1-L-CKO lenses. Mature lenses of Foxp1-L-CKO mice were small and opaque. The fiber cell structure in the core and the cortical fiber cell columns were disturbed in Foxp1-L-CKO mice at postnatal day 14, potentially accounting for the opacity. In addition, epithelial cells were not aligned into columns along the transition zone in Foxp1-L-CKO mice. Taken together, these results suggest that Foxp1 has a role during lens growth in epithelial and differentiating fiber cells. Conclusions: Loss of Foxp1 results in loss of suture and fiber cell alignment, which eventually causes lens opacity, suggesting that Foxp1 has a key role in establishing cortical lens architecture.

Transition of differential histone H3 methylation in photoreceptors and other retinal cells during retinal differentiation.

To analyze cell lineage-specific transitions in global transcriptional and epigenetic changes during retinogenesis, we purified retinal cells from normal mice during postnatal development into two fractions, namely, photoreceptors and other retinal cells, based on Cd73 expression, and performed RNA sequencing and ChIP sequencing of H3K27me3 and H3K4me3. Genes expressed in the photoreceptor lineage were marked with H3K4me3 in the Cd73-positive cell fraction; however, the level of H3K27me3 was very low in both Cd73-positive and -negative populations. H3K27me3 may be involved in spatio-temporal onset of a subset of bipolar-related genes. Subsets of genes expressed in amacrine and retinal ganglion cells, which are early-born retinal cell types, were suggested to be maintained in a silent state by H3K27me3 during late-stage retinogenesis. In the outer nuclear layer, upregulation of Rho and rod-related genes were observed in Ezh2-ablated retina, suggesting a role for H3K27me3 in the maintenance of proper expression levels. Taken together, our data on the transition of lineage-specific molecular signatures during development suggest that histone methylation is involved in retinal differentiation and maintenance through cell lineage-specific mechanisms.

BMP signaling participates in late phase differentiation of the retina, partly via upregulation of Hey2.

Bone morphogenetic protein (BMP) plays pivotal roles in early retinal development. However, its roles in the late phase of retinal development remain unclear. We found that BMP receptors and ligands were expressed in the postnatal mouse retina. Furthermore, immunostaining revealed that phosphorylated Smads were enriched in various cells types in the inner nuclear layer postnatally. However, phosphorylated Smads were not detected in photoreceptors, suggesting that BMP may play roles in retinal cells in the inner nuclear layer. Forced expression of constitutively active BMP receptors during retinal development resulted in an increased number of bipolar cells and Müller glia and a decreased number of rod photoreceptors; however, proliferation was not perturbed. The expression of dominant negative BMP receptors resulted in a decreased number of Müller glia and bipolar cells. In addition, inhibiting BMP signaling in retinal monolayer cultures abrogated Müller glial process extension, suggesting that BMP signaling also plays a role in the maturation of Müller glia. The expression of the basic helix-loop-helix transcription factor Hey2 was induced by BMP signaling in retinas. The coexpression of sh-Hey2 with constitutively active BMP receptors suggested that the effects of BMP signaling on retinal differentiation could be attributed partly to the induction of Hey2 by BMP. We propose that BMP signaling plays pivotal roles in the differentiation of retinal progenitor cells into late differentiating retinal cell types and in the maturation of Müller glia; these effects were mediated, at least in part, by Hey2.