The Rax homeoprotein in Müller glial cells is required for homeostasis maintenance of the postnatal mouse retina

Müller glial cells, which are the most predominant glial subtype in the retina, play multiple important roles, including the maintenance of structural integrity, homeostasis, and physiological functions of the retina. We have previously found that the Rax homeoprotein is expressed in postnatal and mature Müller glial cells in the mouse retina. However, the function of Rax in postnatal and mature Müller glial cells remains to be elucidated. In the current study, we first investigated Rax function in retinal development using retroviral lineage analysis and found that Rax controls the specification of late-born retinal cell types, including Müller glial cells in the postnatal retina. We next generated Rax tamoxifen–induced conditional KO (Rax iCKO) mice, where Rax can be depleted in mTFP-labeled Müller glial cells upon tamoxifen treatment, by crossing Raxflox/flox mice with Rlbp1-CreERT2 mice, which we have produced. Immunohistochemical analysis showed a characteristic of reactive gliosis and enhanced gliosis of Müller glial cells in Rax iCKO retinas under normal and stress conditions, respectively. We performed RNA-seq analysis on mTFP-positive cells purified from the Rax iCKO retina and found significantly reduced expression of suppressor of cytokine signaling-3 (Socs3). Reporter gene assays showed that Rax directly transactivates the Socs3 promoter. We observed decreased expression of Socs3 in Müller glial cells of Rax iCKO retinas by immunostaining. Taken together, the present results suggest that Rax suppresses inflammation in Müller glial cells by transactivating Socs3. This study sheds light on the transcriptional regulatory mechanisms underlying retinal Müller glial cell homeostasis.Yoshimoto T., Chaya T., Varner L.R., et al. The Rax homeoprotein in Müller glial cells is required for homeostasis maintenance of the postnatal mouse retina. Journal of Biological Chemistry 299, 105461 (2023); https://doi.org/10.1016/j.jbc.2023.105461

The potential role of Arhgef33 RhoGEF in foveal development in the zebra finch retina

The fovea is a pit formed in the center of the retina that enables high-acuity vision in certain vertebrate species. While formation of the fovea fascinates many researchers, the molecular mechanisms underlying foveal development are poorly understood. In the current study, we histologically investigated foveal development in zebra finch (Taeniopygia guttata) and found that foveal pit formation begins just before post-hatch day 14 (P14). We next performed RNA-seq analysis to compare gene expression profiles between the central (foveal and parafoveal) and peripheral retina in zebra finch at P14. We found that the Arhgef33 expression is enriched in the middle layer of the inner nuclear layer at the parafovea, suggesting that Arhgef33 is dominantly expressed in Müller glial cells in the developing parafovea. We then performed a pull-down assay using Rhotekin-RBD and observed GEF activity of Arhgef33 against RhoA. We found that overexpression of Arhgef33 in HEK293 cells induces cell contraction and that Arhgef33 expression inhibits neurite extension in Neuro 2A cells, which is partially recovered by a Rho-kinase (ROCK) inhibitor. Taken together, we used zebra finch as a model animal to investigate foveal development and identified Arhgef33 as a candidate protein possibly involved in foveal development through modulating RhoA activity

Analysis of Transcriptional Regulatory Pathways of Photoreceptor Genes by Expression Profiling of the Otx2-Deficient Retina

In the vertebrate retina, the Otx2 transcription factor plays a crucial role in the cell fate determination of both rod and cone photoreceptors. We previously reported that Otx2 conditional knockout (CKO) mice exhibited a total absence of rods and cones in the retina due to their cell fate conversion to amacrine-like cells. In order to investigate the entire transcriptome of the Otx2 CKO retina, we compared expression profile of Otx2 CKO and wild-type retinas at P1 and P12 using microarray. We observed that expression of 101- and 1049-probe sets significantly decreased in the Otx2 CKO retina at P1 and P12, respectively, whereas, expression of 3- and 4149-probe sets increased at P1 and P12, respectively. We found that expression of genes encoding transcription factors involved in photoreceptor development, including Crx, Nrl, Nr2e3, Esrrb, and NeuroD, was markedly down-regulated in the Otx2 CKO at both P1 and P12. Furthermore, we identified three human retinal disease loci mapped in close proximity to certain down-regulated genes in the Otx2 CKO retina including Ccdc126, Tnfsf13 and Pitpnm1, suggesting that these genes are possibly responsible for these diseases. These transcriptome data sets of the Otx2 CKO retina provide a resource on developing rods and cones to further understand the molecular mechanisms underlying photoreceptor development, function and disease

ICKは細胞種特異的な繊毛形成と繊毛内輸送の制御に必須である

京都大学0048新制・課程博士博士(医学)甲第18507号医博第3927号新制||医||1005(附属図書館)31393京都大学大学院医学研究科医学専攻(主査)教授 渡邉 大, 教授 近藤 玄, 教授 斎藤 通紀学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDFA

Role of the mouse retinal photoreceptor ribbon synapse in visual motion processing for optokinetic responses.

The ribbon synapse is a specialized synaptic structure in the retinal outer plexiform layer where visual signals are transmitted from photoreceptors to the bipolar and horizontal cells. This structure is considered important in high-efficiency signal transmission; however, its role in visual signal processing is unclear. In order to understand its role in visual processing, the present study utilized Pikachurin-null mutant mice that show improper formation of the photoreceptor ribbon synapse. We examined the initial and late phases of the optokinetic responses (OKRs). The initial phase was examined by measuring the open-loop eye velocity of the OKRs to sinusoidal grating patterns of various spatial frequencies moving at various temporal frequencies for 0.5 s. The mutant mice showed significant initial OKRs with a spatiotemporal frequency tuning (spatial frequency, 0.09 ± 0.01 cycles/°; temporal frequency, 1.87 ± 0.12 Hz) that was slightly different from the wild-type mice (spatial frequency, 0.11 ± 0.01 cycles/°; temporal frequency, 1.66 ± 0.12 Hz). The late phase of the OKRs was examined by measuring the slow phase eye velocity of the optokinetic nystagmus induced by the sinusoidal gratings of various spatiotemporal frequencies moving for 30 s. We found that the optimal spatial and temporal frequencies of the mutant mice (spatial frequency, 0.11 ± 0.02 cycles/°; temporal frequency, 0.81 ± 0.24 Hz) were both lower than those in the wild-type mice (spatial frequency, 0.15 ± 0.02 cycles/°; temporal frequency, 1.93 ± 0.62 Hz). These results suggest that the ribbon synapse modulates the spatiotemporal frequency tuning of visual processing along the ON pathway by which the late phase of OKRs is mediated

Obif, a Transmembrane Protein, Is Required for Bone Mineralization and Spermatogenesis in Mice

<div><p>Background</p><p>Various kinds of transmembrane and secreted proteins play pivotal roles in development through cell-cell communication. We previously reported that <i>Obif</i> (Osteoblast induction factor, Tmem119), encoding a single transmembrane protein, is expressed in differentiating osteoblasts, and that <i>Obif^−/−</i> mice exhibit significantly reduced bone volume in the femur. In the current study, we characterized the Obif protein and further investigated the biological phenotypes of a variety of tissues in <i>Obif^−/−</i> mice.</p><p>Results</p><p>First, we found that O-glycosylation of the Obif protein occurs at serine residue 36 in the Obif extracellular domain. Next, we observed that <i>Obif^−/−</i> mice exhibit bone dysplasia in association with significantly increased osteoid volume per osteoid surface (OV/OS) and osteoid maturation time (Omt), and significantly decreased mineral apposition rate (MAR) and bone formation rate per bone surface (BFR/BS). In addition, we observed that <i>Obif^−/−</i> mice show a significant decrease in testis weight as well as in sperm number. By histological analysis, we found that <i>Obif</i> is expressed in spermatocytes and spermatids in the developing testis and that spermatogenesis is halted at the round spermatid stage in the <i>Obif^−/−</i> testis that lacks sperm. However, the number of litters fathered by male mice was slightly reduced in <i>Obif^−/−</i> mice compared with wild-type mice, although this was not statistically significant.</p><p>Conclusions</p><p>Our results, taken together with previous observations, indicate that Obif is a type Ia transmembrane protein whose N-terminal region is O-glycosylated. In addition, we found that <i>Obif</i> is required for normal bone mineralization and late testicular differentiation <i>in vivo</i>. These findings suggest that <i>Obif</i> plays essential roles in the development of multiple tissues.</p></div

Histological analysis of distal femoral epiphysis of <i>Obif</i>^−/− mice.

<p><b>(A-B)</b> Villanueva bone staining of distal femur sections from wild-type and <i>Obif</i>^−/− mice. The thickness of distal femoral growth plates was unaltered between wild-type (white box) and <i>Obif</i>^−/− mice (black box) <b>(A)</b>. In wild-type and <i>Obif</i>^−/− mice, the osteoblasts (indicated by arrowheads) and osteoclasts (indicated by arrows) were unchanged in number and size. Scale bars represent 100 μm <b>(A)</b> and 20 μm <b>(B)</b>. Error bars show the SEM (n = 3).</p

<i>Obif</i> is required for normal spermatogenesis.

<p><b>(A)</b> Gross appearance of testes in male wild-type and <i>Obif</i>^−/− mice at 12 wks. <b>(B)</b> Comparison of testicular weight in wild-type (white box), <i>Obif</i>^+/− (grey box), and <i>Obif</i>^−/− (black box) mice at 5 wks and 12 wks (n = 6). <b>(C)</b> Comparison of epididymis weight between wild-type and <i>Obif</i>^−/− mice at 12 wks (n = 6). <b>(D)</b> Comparison of sperm number from cauda epididymis between wild-type, <i>Obif</i>^+/−, and <i>Obif</i>^−/− at 16 wks and 24 wks (n = 4). <b>(E)</b> The level of serum testosterone in wild-type and <i>Obif</i>^−/− mice at 12 wks (n = 6). <b>(F)</b> H&E staining of testis sections from wild-type and <i>Obif</i>^−/− mice at 12 wks. Scale bars represent 5 mm (<b>A</b>), and 50 μm <b>(F)</b>. Error bars show the SEM. *P < 0.05.</p

Obif protein is O-glycosylated at serine residue 36.

<p><b>(A)</b> Potential O-glycosylation sites of human and mouse Obif proteins. The predicted amino acid sequences of human OBIF (NP_859075.2) and mouse Obif (NP_666274.1) were aligned by the ClustalW program (<a href="http://clustalw.ddbj.nig.ac.jp/" target="_blank">http://clustalw.ddbj.nig.ac.jp/</a>). Asterisks indicate identical amino acids. Colons and periods indicate similar amino acids. Yellow boxes indicate potential O-glycosylation sites. Red bracket indicates extracellular domain. Blue underline indicates N-terminal signal peptides. Green box indicates single transmembrane domain. (<b>B-C</b>) Analysis of O-glycosylation sites in the mouse Obif protein. Constructs of pCAGGS expression vector (CAG), FLAG-tagged GFP (GFP), or FLAG-tagged mObif with or without mutation(s) (wild-type (WT), S36A, S43A, T54A, T60A, T67A, or S36A/S43A/T54A/T60A/T67A (ALL)) were transfected into HEK293T cells. The HEK293T cells were cultured for 24 h. The cell lysates were analyzed by Western blotting using an anti-FLAG M2 antibody <b>(B)</b>. FLAG-tagged constructs expressing GFP (GFP), human CD55 (hCD55), or wild-type mouse Obif (mObif-WT) were transfected into HEK293T cells cultured in standard medium. The HEK293T cells were cultured for 24 h, and subsequently cultured for 3 days in medium with or without benzyl-GalNAc. The cell lysates were analyzed by Western blot analysis using the anti-FLAG M2 antibody <b>(C)</b>. Arrowheads indicate the 60 kDa band of O-glycosylated mObif. Arrows indicate the 37 kDa band is a nascent form of mObif-WT. Benzyl-GalNAc, benzyl 2-acetamido-2-deoxy- α-D-galactopyranoside, an O-glycosylation inhibitor.</p